Because of the high incidence of adverse reactions to standard anti- Pneumocystis carinii chemotherapeutic agents, and the failure of such agents in 10-30% of AIDS patients with Pneumocystis Carinii pneumonia, the development of effective alternative therapeutic agents is needed. Standard drug screening has relied on the use of an animal model, which is expensive and cumbersome, and thus in vitro cultures systems are needed. Based on earlier studies, we have developed two short-term in vitro culture systems. The first is based on metabolism by Pneumocystis carinii of paraminobenzoic acid (PABA). The second is based on incorporation by P. carinii of radio labeled methionine into P. carinii proteins, specifically, the major surface glycoprotein of P. carinii. The latter assay is more rapid and allows screening of larger numbers of compounds. Using the latter assay, we have previously demonstrated that a selective DHFR inhibitor provided by Roche Pharmaceuticals is very active against P. carinii in vitro. This agent has subsequently been demonstrated by others to have activity in an animal model of P. carinii pneumonia. Although over the past year we have not screened any new agents, we are maintaining the animal model of P. carinii pneumonia so that as potential agents are identified, either by ourselves or in collaboration with drug companies or other institutes, we will be able to screen such agents. The assays that we have developed should allow a more rapid screening of a large number of chemotherapeutic agents and at the same time, will reduce the number of animals that need to be utilized in drug screening studies. The ultimate goal is to develop drugs that are more effective or less toxic than those that are currently available for treating Pneumocystis carinii pneumonia.